Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver.
We have investigated the putative carbamylphosphate- and ornithine-binding domains in ornithine transcarbamylase from rat liver using site-directed mutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-X and therefore implicated in the binding of the phosphate moiety of carbamylphosphate has been replaced with a leucine. This results in a dramatic reduction of catalytic activity, although the enzyme is synthesized in cells stably transfected with the mutant clone and imported, correctly processed and assembled into a homotrimer in mitochondria. The sole cysteine residue (Cys271) has been implicated in ornithine binding by the chemical modification studies of Marshall and Cohen in 1972 and 1980 (J. Biol. Chem., 247, 1654-1668, 1669-1682; 255, 7291-7295, 7296-7300). Replacement of this residue with serine did not eliminate enzyme activity but affected the Michaelis constant for ornithine (Kb), increasing it 5-fold from 0.71 to 3.7 mM and reduced the kcat at pH 8.5 by 20-fold. These changes represent a loss in apparent binding energy for the enzyme--ornithine complex of 2.9 kcal/mol, suggesting that Cys271 is normally involved in hydrogen bonding to the substrate, ornithine. The cysteine to serine substitution also caused the dissociation constant (Kii) for the competitive inhibitor, L-norvaline to be increased 10-fold, from 12 to 120 microM. The small loss in binding energy and relatively high residual catalytic activity of the mutant strongly suggests that a number of other residues are involved in the binding of ornithine.(ABSTRACT TRUNCATED AT 250 WORDS)
McDowall
S
S
Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
van Heeswijck
R
R
Hoogenraad
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism; D005354:Fibrosarcoma; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D002221:Carbamyl Phosphate; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase